Volume 2, Number 2, August 2008

p 73 - 78

ISSN 1978-3477

Optimum Concentration of Glucose and Orange II for Growth and

Decolorization of Orange II by Enterococcus faecalis ID6017
under Static Culture
V. IRENE MEITINIARTI1*, ENDANG S. SOETARTO2, EKO SUGIHARTO3,

AND

KRIS HERAWAN TIMOTIUS1

Faculty of Biology, Universitas Kristen Satya Wacana, Jalan Diponegoro 52-60, Salatiga 50711, Indonesia;
2
Faculty of Biology, 3 Department of Chemistry, Faculty of Natural Science and Mathematics,
Universitas Gadjah Mada, Jalan Teknika Selatan, Sekip Utara, Kampus Bulaksumur, Yogyakarta 55281, Indonesia
Growth and decolorization performance of bacterial grown on azodyes-containing-medium is influenced by various
concentrations of carbon sources and azodyes. The optimum level of glucose and Orange II concentration for growth and
Orange II decolorization by Enterococcus faecalis ID6017 are reported in this paper. The experiments were carried out in liquid
static culture as batch experiments. Glucose and Orange II concentrations used in these experiments were 0.45, 0.90, 1.80 g l -1 ,
and 40, 80, 120 mg l-1, respectively. The specific growth rate and decolorization rate of Orange II by E. faecalis were highest
on the medium which contained at least 0.90 g l-1 glucose. It is necessary to note that glucose above 0.90 g l-1 gave no significant
difference. On the medium containing 0.90 g l-1 glucose and 80 mg l-1 Orange II, E. faecalis grew with the highest specific growth
rate (0.28 h-1) and Orange II decolorization rate (0.47 h-1). The maximum specific growth rate of biomass (max) and the halfsaturation coefficient (K S) under optimal conditions were 0.25 h-1 and 1.5 g.l-1, respectively. The kinetics of decolorization
indicated that the process followed first order kinetics with respect to the initial concentration of Orange II. The inhibition
constant (K I) was found to be 750 mg l-1 Orange II, indicating that Orange II concentration at e 750 mg l -1 would inhibit
bacterial growth to decolorize Orange II.

Key words: Enterococcus faecalis, azodyes, decolorization, glucose, Orange II

_____________________________________________
Orange II is one of synthetic azodyes which has been
widely used for coloring of textiles, food, and cosmetics.
Orange II (Acid-Orange-7 or p-(2-hydroxy-1-naphthylazo)
benzenesulfonic acid sodium salt) has the molec(ular
structure C16H11N2NaO4S. 5H20 (Fig 1: Merck Index 1968).
This azodye has been used as a model substrate for azodye
degradation. Orange II can be degraded by Sphingomonas
sp. 1CX and E. faecalis ID6017 into two main intermediate
products i.e. sulphanilic acid and 1-amino-2-naphthol
(Coughlin et al. 1999; Meitiniarti et al. 2007). Both of them
may be mineralized further.
Azodyes are relatively resistant to microbial degradation
under conditions normally found in waste-water treatment
plants. However, several microorganisms are reported to be
able to transform azodyes into non-colored products or even
to mineralized products (Stolz 2001). Recently, a number of
studies have been focusing on microbial degradation of
azodyes (Kim et al. 1995). This is because physicochemical
methods used for color removal of the effluents show
disadvantages in terms of operational problems, high cost
and sludge production (Kapdan et al. 2000; Kodam et al.
2005). According to Rafii et al. (1990), human intestinal
OH
N=N

SO3Na.5H2O

Fig 1 Chemical structrure of Orange II.

________________________
*
Corresponding author, Phone: +62-298-321212,
Fax: +62-298-321433, E-mail: irene_meiti@yahoo.com

microbiota generally found in the effluent of waste water

treatment and using of these bacteria for azodyes
degradation has been reported. In industrial waste-water
containing various dyes, azodye degraders should exhibit
decolorizing ability for a wide range of dyes and
environmental conditions (Fang et al. 2005). In regard to
this E. faecalis is a bacterial species which has been reported
to decolorize azodyes (Handayani et al. 2007). Therefore the
decolorization of azodyes is an important topic for study.
Bacterial azodyes degradation is usually initiated by
cleavage of the azo bond under anaerobic conditions
resulting in the formation of aromatic amine colorless
products. Hence the process is called decolorization
(Zimmermann et al. 1982; Tan 2001; van der Zee 2002).
The performance of microbial decolorization rates
depends on the activity of the microorganism through a
specific enzymatic process and is affected by environmental
conditions, such as substrate concentration, composition
of the medium, pH, carbon and nitrogen sources, and
incubation temperature. Sphingomonas sp. strain 1CX has
an ability to decolorize 20 mg l-1 Orange II, Acid Orange 8,
Acid Orange 10, Acid Red 4, or Acid Red (Coughlin et al.
1997), where as E. faecalis ID6017 has an ability to decolorize
Acid Red 27 and Reactive Red 2 up to concentration of
100 mg l-1 in batch system (Handayani et al. 2007) and
Orange II up to concentration 120 mg l-1 in fedbacth system
(Meitiniarti et al. 2006). Meitiniarti et al. (2005) reported that
E. faecalis produces an Orange II reductase. Several
environmental conditions, such as substrate and cosubstrate concentration, could influence the enzymes activity
(Tan 2001). In this paper, we discuss the optimum level of
glucose as co-substrate and Orange II concentrations on
the growth kinetics of E. faecalis and Orange II decolorization
performance.

74 MEITINIARTI

ET AL.

Microbiol Indones

MATERIALS AND METHODS

Microorganism and Culture Condition. E. faecalis
ID6017 was used as a bacterial model for the decolorization
of Orange II and was maintained on a slant media of
Trypticase Soy Agar (TSA) at room temperature (26-28C)
as a stock culture. E. faecalis was grown on liquid-basalmedium consisting of (g l -1): 0.25 MgSO 4.7H2 O, 1.98
(NH4)2SO4, 5.55 K2HPO4, 2.13 KH2PO4, and 0.25 yeast extract.
Glucose was added in concentrations of 0.45, 0.90, and
1.80 g l-1 Orange II (Merck, CI Acid Orange 7, CI 15510,
MW= 440.41) was added in concentrations of 0, 40, 80, and
120 mg l-1.
Inoculum Preparation. 48-h-old TSA slant cultures of
E. faecalis were grown on 500 ml Erlenmeyer flask, containing
basal medium without Orange II, for 24 h on a shaker (150 rpm).
This culture was the pre-culture (inoculum).
Experimental Design and Culture Conditions. The
experiments were carried out in two batchs using liquid
medium for 9 h at room temperature. In the first experiment,
three flasks each containing 80 mg l-1 Orange II medium were
prepared and each flask was added 0.45, 0.90, and 1.80 g l-1
glucose. This experiment was done to determine the glucose
concentration that increased bacterial growth and Orange II
decolorization rate. The glucose concentration which resulted
in increasing growth and decolorization rate, was selected
and used as the limited-substrate-concentration. In the
second group of experiments, the selected glucose
concentration was then supplemented with 40, 80, and
120 mg l-1 Orange II concentrations. Experiments was carried out in triplicate.
As much as 50 ml aliquots of pre-culture were transfered
to 450 ml medium in a 1 l volume growing vessels (Duran)
with a rubber stopper, and incubated under static conditions at room temperature. At interval of one hour, 10 ml
aliquots of each culture was removed up the late exponential
growth-phase. Parameters examined were concentrations of
glucose, Orange II, cell mass (biomass) and sulphanilic acid
(as a decolorization product of Orange II).
Analytical Methods. Samples were centrifused at 3 326g
for 30 min to separate biomass from the supernatant. The
supernatant was analyzed for glucose, Orange II, and
sulphanilic acid concentrations. After being washed twice,
the biomass was re-suspended into an initial volume (10 ml)
using distilled water and examined spectrophotometrically
( 600nm). The biomass concentration in units of cell dry weight
was calculated using a standard curve of optical density
(OD600nm; y-axis) against biomass concentration (cell dry
weight) (mg l-1; x-axis).
Glucose concentrations were determined spectrophotometrically ( 540nm) using the DNS reagent method
(James 1995) and Orange II concentration were determined
spectrophotometrically ( 482nm) (Zimmermann et al. 1982).
The sulphanilic acid concentration was analyzed by
injecting five to ten l sampels of supernatant into a HPLC
Shimadzu model LC-3A chromatograph, equipped with UVdetector and ODS column (150 mm x 8 mm). The mobile phase
was composed of methanol and acetic acid 0.6% (v/v) = 60:40.
The flow rate was 0.7 ml min-1. The eluent were monitored

using a UV detector at 276 nm (Chang et al. 2001; Supaka et

al. 2004).
Determination of Kinetic Parameters (k, , max, KS, and
KI). The ability of bacterial strains to decolorize Orange II
was determined by calculating the decolorization rate used
equation (1) (Wuhrmann et al. 1980):
ln (C0-C)
k =
(t-t 0)
Note: k is decolorization rate (decrease of color intensity);
C0 and C are color concentrations (mg l-1) at t0 and t; t0 and t
are time (h).
The substrate/co-substrate consumption and decreasing
of decolorization product (sulphanilic acid) was calculated
using equation (2):
S = S0-St

or S = St-Se

Note: S is substrate/co-substrate consumption (mg); S0 is

glucose/Orange II concentration (mg l-1) at t0; St is the lowest
glucose/Orange II concentration or the highest sulphanilic
acid concentration (mg l-1); Se is the lowest sulphanilic acid
concentration (mg l-1); t0, t, and te are time (h).
The specific growth rate () is determined from the cell
mass as a first order with the following equation:

dX
= X , X = X 0 at t=0
dt
Integration of equation 3 yields:

ln

X
= t , or X = X 0e t
X0

and X0 are biomass concentrations at time t and

t=0.
Generally, the growth rate is restricted by the concentration
of the growth-limiting substrate that can be described by the
Monod Equation:

maxwhere X

max .S
KS + S

where max is the maximum specific growth rate when S>>KS.

The constant KS is the saturation constant or half-velocity
constant and is equal to the concentration of the ratelimiting substrate when the specific rate of growth is equal
to 50% of the maximum.
In this paper, the Monod equation modified by the Hanes
model (called the Hanes-Monod Model) was used to analyze
the values of max and KS (Brandt 2001):

max

KS

max

A plot of against is linear with a slope of 1/u and an

intercept on the y-axis of

When a substrate is inhibited by its own biodegradation,

like Orange II, the Monod model was derivatived by
incorporating the inhibition constant KI. Among the substrate
inhibition models, the Andrews equation (7) is most widely
used (Okpokwasili and Nweke 2005):

Volume 2, 2008

Microbiol Indones 75

= max

KS + S +

S2
KI

where KI is the inhibition constant (calculated by Microsoft

Excel Software in this paper).
RESULTS
Optimal Level of Glucose. The addition of various
concentration of glucose (0.45 to 1.80 g l-1) did not give
any significant difference in the specific growth rate, biomass
production of E. faecalis or the concentration of sulphanilic
acid (Table 1 and Fig 2). However, as shown in Table 1, the
decolorization of Orange II was significantly influenced by
the addition of glucose. By calculating glucose consumption
in three initial glucose concentrations, we showed that by
increasing the glucose concentration, from 0.90 to 1.80 g l-1, this
did not efficiently increased the specific growth rate, biomass
production, and Orange II reduction by E. faecalis. Thus, in
the second experiment, we used optimal glucose concentration
i.e. 900 mg l-1.
Optimal Level of Orange II. The specific growth rate of
E. faecalis in Orange II concentration from 40 to 120 mg l-1
the optimal glucose concentration was not significantly
different to that of the control (Fig 3). Based on the specific
growth rate of E. faecalis, our results shown that by
increasing the Orange II concentration did not increase the
specific growth rate. By calculating the kinetic parameter of
growth our results show that the best specific growth rate of

E. faecalis was on the 80 mg l-1 Orange II-containingmedium (Table 2). In the Orange II 120 mg l-1 containing
medium, the decolorization rate and capacity were higher
than the lower Orange II concentration (40-80 mg l-1) (Table 2).
The Maximum Specific Growth Rate (max), the HalfMaximum Saturation Coefficient (Ks), and the Inhibition
Constant (KI). By calculating max and Ks values using the
Hanes-Monod model for E. faecalis growing on medium
containing various glucose consentrations, we obtained
values of max and Ks of 0.22 h-1 and 0.05 g l-1, respectively
(Fig 4). By similar methods, the values of max and Ks for
E. faecalis growth on medium containing various Orange II
concentrations could be determined. The values of max and
Ks were 0.25 h-1 and 1.5 mg l-1, respectively (Fig 5). Based on
the kinetic parameters obtained: m (0.26, 0.28, and 0.25 h-1 for
40, 80, and 120 mg l-1 Orange II, respectively), max (0.25),
and Ks (1.5) with various initial S (Orange II) concentrations,
the KI value could be calculated using microsoft excel
programme by inserting the assumed KI value as a variable.
After 15 iterations, the best fit (i.e between the m values
calculated by Hanes-Monod method and the m Haldane,
correlation coefficient R2 1) gave an Orange II KI value of
750 mg l-1.
DISCUSSION
As shown by the data in Table 1 and Fig 2, increasing the
glucose concentration from 0.45 to 0.90 g l-1, increased
specific growth rate and biomass production. However,
increasing the glucose concentration from 0.90 to 1.80 g l-1
did not significantly increase the specific growth rate and

Table 1 The influence of glucose on Enterococcus faecalis ID6017 growth and its performance for Orange II decolorization in the medium
containing 80 mg l-1 Orange II and several concentrations of glucose
Glucose concentration (g l-1)
0.45
0.90
0.18 + 0.01
0.20 + 0.02
29.3 + 2.518.04*
30.1 + 7.422.86*
0.39 + 0.005
0.67 + 0.007
65.9 + 4.4
71.1 + 3.2
0.51 + 0,1
0.78 + 0.2
3.65 + 0.3
3.11 + 0.1
32.5 + 0.3
35.5 + 0.12

Parameter of growth and decolorization

Specific growth rate (h-1)
Biomass produced (mg)
Glucose consumption (g)
Orange II reduction (mg)
Decolorization rate (h-1)
Capacity of decolorization (mg substrate/mg cell d.w.)
Concentration of sulphanilic acid (mg l-1)
*biomass produced during decolorization of Orange II.

b
50
45
40
35
30
25
20
15
10
5
0

y2= 12.628e0.2042x

Ln Orange II concn (mg l-1)

Biomass concn (mg l-1)

R = 0.9513
y1= 13.091e0.1797x
R2= 0.991
y3= 8.8155e0.2086x
R2= 0.9845

5
4.5
4
3.5
3
2.5
2

y1= -0.5039x + 5.3186

R 2= 0.9479

y3= -0.7782x + 6.1293

R2= 0.9666

1.5
1
0.5
0

y2= -0.7832x + 6.1398

R2= 0.9616
0

Incubation time (h)

(

1.80
0.21 + 0.02
31.3 + 3.716.52*
0.79 + 0.009
72.5 + 8.6
0.77 + 0.2
4.39 + 0.5
36.7 + 0.71

) 1= 0.1797 h-1; (

) 2= 0.2042 h-1; (

) 3= 0.2086 h-1

) k1= 0.509 h-1; (

3
4
Incubation time (h)

) k2= 0.7832 h-1; (

) k3= 0.7782 h-1

Fig 2 Specific growth rate (a) and Orange II decolorization rate, (b) during Enterococcus faecalis growing on the medium containing
Orange II and 0.45 g l-1 glucose ( ), 0.90 g l-1 glucose ( ), and 1.80 g l-1 glucos ( ).

76 MEITINIARTI

ET AL.

Microbiol Indones

Biomas concn (mg l-1)

80
60

40
20
0
0

10

Incubation time (h)

Fig 3 The growth of Enterococcus faecalis on the medium without Orange II ( ), with 40 mg l-1; ( ), 80 mg l-1; ( ), and 120 mg
l-1 ( ), Orange II. Bars indicated standard error where P=0.05.

In contrast to the influence of glucose on bacterial

growth, the decolorization of Orange II was significantly
influenced by the addition of glucose. The consumption of
glucose was related with the rate of decolorization and the
amount of reduced Orange II. This means that additional
glucose as an energy source is needed by the E. faecalis to
decompose Orange II i.e. decolorization of Orange II is an
energy consuming activity.
The addition of glucose did not give any significant
difference on sulphanilic acid formation. Based on the
concentration of sulphanilic acid at the end of cultivation, it
showed that the concentration of sulphanilic acid was quite
high. This is because this bacteria can not metabolise
sulphanilic acid (Meitiniarti et al. 2007), although the added

Table 2 The influence of Orange II on Enterococcus faecalis ID6017 growth and its performance for Orange II decolourization in the
medium contained 0.90 g l-1 glucose and several concentrations of Orange II
Parameter of growth and decolorization
Specific growth rate (h-1)
Biomass produced (mg)

0
0.28 + 0.03
51.77 + 8.10

Glucose consumption (g)

695.6 + 12.9

Orange II reduction (mg)

Orange II reduction (%)

n.d

Decolorization rate (h -1)

Dye removal capacity (mg substrate/mg cell d.w)
Concentration of sulphanilic acid (mg l-1)

n.d
n.d
n.d

Orange II concentration (mg l-1)

40
80
0.26 + 0.02
0.28 + 0.02
45.04 + 11.90
34.1 + 7.1
25.10*
22.9*
718.2 + 38.6
732.7 + 25.0

120
0.25 + 0.03
33.72 + 7.50
23.90*
756.2 + 22.9

33.63 + 4.70
84.00
0.95 + 0.14
1.55 + 0.19
24.2 + 2.9

74.45 + 6.38
62.00
0.32 + 0.05
2.77 + 0.57
37.02 + 0,71

64.22 + 6.38
80.27
0.47 + 0.07
2.64 + 0.32
34.02 + 3.09

10
8

y= 4.6111x + 0.2503
R2= 0.9961

6
4
2
0
0

0.5
1.0
Glucose concentration (S) (g l-1)

1.5

2.0

Fig 4 A plot of glucose concentration/specific growth rate (S/)

vs glucose concentration (S) resulting in a straight line with slope
equal to 1/max= 4.61 and an intercept of KS/max= 0.25 for various cosubstrate concentrations on growing medium of Enterococcus faecalis.

biomass produced by the bacteria. This might be because

the glucose concentration of 0.90 g l-1 is adequate to support
the growth of E. faecalis. It was also shown from glucose
consumption and biomass production that these were low
during bacterial growth. For their growth, bacteria require C
and N sources in certain proportions (Thingstad and Lignell
1997). In the 0.90 g l-1 glucose containing medium, the
proportions of C and N sources were suitable for bacterial
growth. Thus, the bacteria grew fast and produced more
biomass in this medium.

Orange II concn/specific growth rate

(S/)

Glucose concn/specific growth rate (S/)

*biomass produced during removal of Orange II, n.d: not determined.

600
500
400
300
200
100
0
0

50
100
150
Orange II concentration (S) (mg l-1)
Fig 5 A plot Orange II concentration/specific growth rate (S/)
vs Orange II concentration (S) resulting in a straight line with a slope
equal to 1/ max= 3.93 and an intercept of KS/ max= 5.89 for various
substrate concentrations on growing medium of Enterococcus faecalis.

amount of glucose was related to the rate of decolorization

and the amount of reduced Orange II.
The increase in Orange II concentration from 40 to
80 mg l-1 raised the specific growth rate of E. faecalis.
However, when Orange II was increased up to 120 mg l -1,
it decreased the specific growth rate of E. faecalis. These
results show that E. faecalis did not use Orange II as a
growth substrate and might be toxic for bacterial growth.
The highest specific growth rate of E. faecalis was on the
medium containing 80 mg l-1 Orange II. This might be due to
the fact that in 80 mg l-1 Orange II concentration, the bacteria

Volume 2, 2008

nutrient requirement was fullfilled and they could tolerate

Orange II concentration. So, they could grow favorably.
Based on the decolorization rate (Table 2), our results
show that the increase in Orange II concentration also
increased the decolorization rate. This is because the
reductase enzyme activity of E. faecalis to remove or reduce
Orange II, was related to substrate concentration (Orange
II). The kinetic analysis of decolorization indicated that the
decolorization process followed first order kinetics with
respect to the initial concentration of Orange II. E. faecalis
had the highest Orange II decolorization rate on the medium
containing 80 mg l-1 Orange II because E. faecalis could
not use Orange II as a growing substrate. At a higher
concentration, Orange II did inhibit E. faecalis growth.
As shown on Table 2, the increase in the decolorization
rate at higher Orange II concentrations would not decrease
sulphanilic acid concentration. It seems that although the
sulphanilic acid concentration increased due to increasing
of Orange II decolorization, E. faecalis could not metabolise
sulphanilic acid. This would result in the accumulation of
sulphanilic acid in the medium. Mendez-Paz et al. (2005)
reported that sulphanilic acid produced by Orange II
decolorization was not degrade futher and it would then
be accumulated in the medium.
The max value obtained from E. faecalis, grown on
medium containing various glucose concentrations, was
similar to the specific growth rate of E. faecalis which was
grown on the medium contained of 0.9-1.8 g l-1 glucose
concentration. This result showed that by increasing the
glucose concentration up to 1.86 g l-1, it would not efficiently
increase the growth of E. faecalis. A low value of Ks obtained
here indicated that bacteria had a very high affinity for
glucose as a carbon substrate. Hence, the bacterial growth
would not be affected until the glucose concentration
declined to a very low level. This result also showed that
using glucose at an optimal concentration is quite economic
when applied in waste-water-treatment plants.
The max values of E. faecalis growth with various
Orange II concentrations was similar with the specific growth
rate of E. faecalis grown on the medium containing lower
than 40 mg l-1 Orange II concentration. This result indicated
that Orange II was not a growth substrate for E. faecalis.
Futhermore, when the Ks value of Orange II was compared
with that of glucose, it showed that bacterial affinity to
Orange II was lower than to glucose. This result indicated
that E. faecalis would preferentially use glucose on the
medium for rapid growth (Thingstad and Lignell 1997).
Although a decrease in Orange II concentration occurred in
these experiments, it is not be due to Orange II consumption
as a substrate. Orange II might be used together with direct
growing supporting substrate via cometabolism.
Based on the value (750 mg l-1) of inhibition coefficient
KI obtained in this experiment, it is shown that Orange II at a
concentration of 750 mg l-1 would inhibit growth of E.
faecalis. The KI value of Orange II also indicated that although
of E. faecalis on the medium contained of 120 mg l-1 Orange II
decreased, Orange II concentration used in this experiment
(40, 80, and 120 mg l-1) was too low to inhibit the growth of E.
faecalis.

Microbiol Indones 77

Based on our results, it can be concluded that by

increasing the glucose (as a co-substrate) concentration up
to 0.90 g l-1 and Orange II up to 80 mg l-1, we could increase
the specific growth rate and Orange II decolorizatin rate of
E. faecalis. An increase in Orange II concentration above
80 mg l-1 would decrease the specific growth rate of E.
faecalis, and when the concentration was increased to more
than 750 mg l-1, the growth of bacteria will be inhibited.
Although the actual dye concentration in effluent is not
more than 750 mg l-1, we can predict that the specific growth
rate of bacteria will decrease when the Orange II concentration
was more than 80 mg l-1. Moreover, our results show that E.
faecalis could not consume sulfanilic acid as one of the
intermediate products of azodye degradation. Accordingly,
it is suggested that this bacteria should not be used alone in
dye-contaminated waste-water treatment, but should be
mixed with other bacterial species which could consume
intermediate products of azodye degradation and had the
ability to degrade other kinds of azodyes.
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